Screening Of Host Proteins Interacted With Mycobacterium Tuberculosis Rv0927c And Identification Of Its Functions

Mycobacterium tuberculosis(M.tb)is an important zoonotic pathogen that causes tuberculosis,which seriously endangers human health and public safety.Tuberculosis is an infectious disease that can be spread through the air,causing about 1.6 million deaths each year.It is the world’s number one killer of infectious diseases.The severe current situation has attracted great attention at home and abroad.In recent years,due to the cross-infection of HIV/TB,the emergence of multidrug-resistant and extensively drug-resistant tuberculosis strains,the uneven protection of BCG vaccines and the increase in population mobility,the prevention and treatment of tuberculosis is facing major challenges.M.tb is an intracellular infection bacterium,and macrophages are its main host cells.In the process of M.tb infection of host cells,M.tb effector protein may interact with host cell proteins,but these interactions mechanism of TB is still unclear,and exploring the interaction and molecular mechanism of M.tb effector protein and host target protein will provide new clues for the prevention and treatment of tuberculosis.Studies have shown that M.tb Rv0927c encodes a short dehydrogenase/reductase and is related to the synthesis of mycolic acid in the cell wall of mycobacteria,and it may be involved in the regulation of host cell inflammatory response.However,the mechanism of Rv0927c function in the process of infecting host cells is still unclear.In order to clarify this mechanism,the following investigations were conducted in this study:1.Screening and verification of host interaction proteins of Mycobacterium tuberculosis Rv0927cIn this study,a total of 26 host target proteins that interact with Rv0927c were screened using the previously constructed macrophage cDNA library and the bait strain of pGBKT7-Rv0927c with yeast two-hybrid method.In order to verify the interaction between Rv0927c and host target protein by co-immunoprecipitation technology,the eukaryotic expression plasmid of Rv0927c and host target protein were constructed with pCMV-HA and pCMV-Myc vectors,the bait plasmid and host target protein particles were co-transfected into HEK293T cells.The whole protein of the cells was collected,IP antibody and Protein A agarose bead were added for immunoprecipitation experiment.The verification results showed that six host proteins interact with Rv0927c,namely Rho GTPase-activated protein 25(ARHGAP25),mitochondrial Tu translation elongation factor(TUFM),chromosome regulatory protein(RCC2),and peptidyl-prolyl isomerase H(PPIH),histone H2B(Hist1h2bf),histone H4(Histone H4).Subsequently,in order to further verify their interaction and investigate intracellular colocalization,fluorescent antibodies were used to label the bait plasmid and the target protein particle respectively and detected by laser confocal experiments.The results showed that ARHGAP25,TUFM and Rv0927c co-localized in the cytoplasm,RCC2 and Rv0927c co-localized in the nucleus and cytoplasm,and PPIH and Rv0927c co-localized in the nucleus.In summary,this study screened and verified four host interaction proteins of Mycobacterium tuberculosis Rv0927c,namely ARHGAP25,TUFM,RCC2 and PPIH.2.Study on the function of Mycobacterium tuberculosis Rv0927c targeting TUFM protein to regulate the host type Ⅰ interferon responseIn this paper,the host protein TUFM was chosen as the target to investigate the function of Mycobacterium tuberculosis Rv0927c to regulate the host type Ⅰ interferon response.Firstly,siRNA silencing was used to interfere with the expression of TUFM in host cells.The results showed that silencing of TUFM result in the up-regulation of the IFN-β in the RAW264.7 cells,indicating that the TUFM molecule negatively regulated type Ⅰ interferon response.Subsequently,endogenous co-immunoprecipitation experiment was performed by transfecting RAW264.7 cells with eukaryotic plasmid pCMV-Rv092 7c.The results showed that Rv0927c and TUFM molecules interacted intracellularly.At the same time,the Rv0927c and TUFM molecular eukaryotic expression plasmids were co-transfected into HEK293T cells,and the mitochondria were fluorescently labeled for laser confocal experiments.The results showed that Rv0927c and TUFM co-localized in intracellular mitochondria.Then,Rv0927c Mycobacterium smegmatis deletion strain rMSΔRv0927c,complementary strain rMSΔRv0927c::Rv0927c,and overexpression strain rMS::pMV261-Rv0927c constructed in the laboratory were used to infect mouse macrophages RAW264.7 with wild strain of Mycobacterium mc2155 as control.The transcription and protein expression levels of IFN-β were measured at different time points after infection by RT-PCR and ELISA.The results showed that Rv0927c can significantly promote the expression of IFN-β.Subsequently,the recombinant strains were used to infect IRF-Luc reporter gene macrophage cell line J774-DualTM,and the activation of type Ⅰ interferon signaling pathway was measured.The results showed that Rv0927c can induce the activation of type Ⅰ interferon signaling pathway.Finally,the Rv0927c recombinant strains were used to infect the cells in which TUFM was silenced by siRNA and the level of IFN-β was measured.The results showed that the expression of IFN-β in the Rv0927c recombinant strains-infected group did not change significantly when TUFM was silenced.These results indicate that Rv0927c targets the host TUFM to regulate type Ⅰ interferon response.Then,the expression of TUFM in RAW264.7 infected by recombinant strain was detected.The results showed that the transcription level and protein level of TUFM significantly increased after infection with Rv0927c-deleted strain,while the transcription level and protein level of the TUFM was significantly down-regulated in RAW264.7 infected by Rv0927c-overexpressed strain.The results indicated that Rv0927c negatively regulate the expression of host TUFM.In summary,this study revealed that Mycobacterium tuberculosis Rv0927c negatively regulates host TUFM by targeting to induce type I interferon expression and activation of type Ⅰ interferon signal pathway.